|DPU: INTERDISCIPLINARY CONFERENCE
|Year : 2020 | Volume
| Issue : 5 | Page : 30-35
Designing novel quercetin derivatives as matrix metalloproteinase-9 inhibitors in colon carcinoma: An In vitro and in silico approach
Sangeeta Ballav1, Kiran Bharat Lokhande2, Ipshita Dabhi3, Sonal Inje1, Amit Ranjan1, K Venkateswara Swamy2, Soumya Basu1
1 Cancer and Translational Research Laboratory, Dr. D. Y. Patil Biotechnology and Bioinformatics Institute, Dr. D. Y. Patil Vidyapeeth, Pune, Maharashtra, India
2 Bioinformatics Research Laboratory, Dr. D. Y. Patil Biotechnology and Bioinformatics Institute, Dr. D. Y. Patil Vidyapeeth, Pune, Maharashtra, India
3 Cancer and Translational Research Laboratory; Bioinformatics Research Laboratory, Dr. D. Y. Patil Biotechnology and Bioinformatics Institute, Dr. D. Y. Patil Vidyapeeth, Pune, Maharashtra, India
|Date of Web Publication||26-Feb-2020|
Cancer and Translational Research Laboratory,
Dr. D. Y. Patil Biotechnology and Bioinformatics Institute, Dr. D. Y. Patil Vidyapeeth, Pune - 411 033, Maharashtra
Source of Support: None, Conflict of Interest: None
Background: Matrix metalloproteinase-9 (MMP-9) plays an important role in activating several signaling molecules and pathways during inflammation, wound healing, and cancer progression. Since flavonoids have been shown to exhibit anti-inflammatory properties, we assessed the antiproliferative activity of quercetin, a flavonoid on colon carcinoma cell line through in vitro assays, and in order to design more specific MMP-9 inhibitors, we have used insilico approach to obtain potent quercetin derivatives. Materials and Methods: The aim of the present study was to investigate the effect of quercetin on cell viability and migration on cultured HCT-15 cells using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay and wound healing assay. The docking analysis of quercetin and its derivatives with the crystal structure of MMP-9 (PDB ID: 3OE9) was performed using FlexX software. Result: Quercetin showed an antiproliferative and antimigratory activity on HCT-15 cells in time- and dose-dependent manner. Conclusion: Novel quercetin derivatives targeting MMP-9 have been identified for further synthesis and validation.
Keywords: Colon cancer, matrix metalloproteinase-9, molecular docking, molecular dynamic simulation, quercetin
|How to cite this article:|
Ballav S, Lokhande KB, Dabhi I, Inje S, Ranjan A, Swamy K V, Basu S. Designing novel quercetin derivatives as matrix metalloproteinase-9 inhibitors in colon carcinoma: An In vitro and in silico approach. J Dent Res Rev 2020;7, Suppl S2:30-5
|How to cite this URL:|
Ballav S, Lokhande KB, Dabhi I, Inje S, Ranjan A, Swamy K V, Basu S. Designing novel quercetin derivatives as matrix metalloproteinase-9 inhibitors in colon carcinoma: An In vitro and in silico approach. J Dent Res Rev [serial online] 2020 [cited 2020 Apr 2];7, Suppl S2:30-5. Available from: http://www.jdrr.org/text.asp?2020/7/5/30/278905
Editor: Dr. Sarika Chaturvedi
| Introduction|| |
In global perspective, colorectal cancer (CRC) is the second most common cancer in women (9.4% of the total cases) and the third most common cancer in men (10.0% of the total cancers). The highest rates are estimated to be in Western Europe, New Zealand, and Australia and the lowest in South-Central Asia and Africa (excluding Southern Africa). Incidence rates differ widely within Asian countries, and the number of CRC cases has increased drastically in certain economically developed parts of Asia. There is an increase of 2–4 times in the incidence of CRC in the past few decades.,, Compared to the Western world, the age-adjusted incidence rates of CRC are low in India as recorded by all cancer registries in India. Notably, with increased urbanization of India, a steady increase in numbers of CRC cases are also increased. Affluent community experiences the rising pattern of incidence and mortality from CRC as compared to the poorer population.
CRC is often diagnosed in advanced stage and associated with metastasis and therapy resistance leading to poor therapeutic outcome. Matrix metalloproteinases (MMPs) are a group calcium-dependent, zinc-containing endopeptidases responsible for the selective proteolytic degradation of most extracellular matrix (ECM) proteins during organogenesis, growth, and normal tissue formation. Degradation of ECM by MMPs is an obligatory step for the migration and invasion of tumor cells and subsequently metastasis. The majority of MMPs are upregulated or disregulated in response to pro-inflammatory cytokines, cell–cell, or cell–ECM interactions. They are also involved in adhesion and migration of the leukocytes from the blood vessels as an inflammatory response. Among all MMPs, MMP-9 is unique for being involved in activating several signaling molecules and pathways during cancer and inflammation, despite being inactive in normal tissues. Overexpression of MMP-9 may lead to poor prognosis in patients with colon cancer. MMP-9, as it is a downstream effector and an upstream mediator of multiple cellular signaling pathways involved in growth and inflammation, has long been foreseen as a promising therapeutic target. It has been observed that inhibition of MMP-9 reduces tumor burden and promotes effector/memory T-cell infiltration and diversity when combined with an anti-PDL1 antibody.
However, despite the presence of strongly promising preclinical data, the overall survival has not been improvised. In addition, MMPs had unforeseen severe side effects. There are few reasons for failure of matrix metalloproteinase inhibitors (MMPIs) in clinical trials. Researchers have utilized semi-selective, nontargeted, broad-spectrum inhibitors targeting MMPs, including MMP-9, few having anti-tumor effects. Broad-spectrum MMPIs may result in tumor progression by blocking these MMPs.
“Exosites,” a molecular structures outside of the catalytic domain, can be targeted with synthetic, low-molecular-weight compounds or antibodies in order to inhibit the MMP activity selectively. Following this approach, the generation of highly selective monoclonal antibodies to MMP-9, AB0041, and AB0046 and the humanized version of AB0041 and GS-5745 were developed with proven efficacy in a mouse xenograft model of colorectal carcinoma.
Flavonoids, a subclass of plant polyphenols, are emerging as promising treatment options for the prevention and treatment of several cancers such as breast, colon, and lung cancers. Recent evidence suggest that in addition to antioxidant properties, flavonoids such as quercetin, apigenin, resveratrol, and kaempferol can directly interact with proteins, making them ideal small molecules for the modulation of cell surface receptors, enzymes, and transcription factors. With an objective to design small molecule inhibitors targeting MMP-9, in this present work, we have assessed the antiproliferative activity of quercetin on colon carcinoma cell line in vitro and also its effect on colon cancer cell migration. Further study was carried out using advanced computational approach such as molecular docking for virtual screening of leads to obtain specific inhibitors of MMP-9, in silico.
| Materials and Methods|| |
3- [4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) dye was purchased from HiMedia. Trypsin solution ([TPEG – 0.25 g trypsin, 0.02 g EDTA, and 0.05 g glucose in 100 ml autoclaved Phosphate buffer saline (PBS) [pH – 7.4]), Roswell Park Memorial Institute (RPMI) 1640, and trypan blue were purchased from HiMedia. Fetal bovine serum (FBS) was procured from Gibco, Thermo Scientific (USA).
Cytotoxicity assay (3- [4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay)
In vitro cell cytotoxicity assay was carried out with HCT-15 by MTT assay. MTT assay is a quantitative colorimetric assay which indicates the proliferation of cells. Cells were maintained with RPMI medium supplemented with 10% FBS in a humid incubator (37°C and 5% CO2) (Eppendorf, Germany). Cells (104 cells/well) were plated onto 96-well tissue culture plates and kept overnight. They were treated with different concentrations of quercetin (10−4, 10−5, 10−6, and 10−7 M) and incubated for 24, 48, and 72 h. 5-flurouracil was used as a positive control. Twenty microliters of the freshly prepared MTT solution (5 mg/ml) was added to each of the well-containing cells incubated for 4 h (37°C and 5% CO2). After incubation, media were removed and 100 μL of dimethyl sulfoxide was added to each well. The viability of cells was determined by measuring their absorbance at 595 nm and 670 nm. The optical density of absorbance is directly proportional to the number of live cells. All the in vitro cytotoxicity experiments were performed in triplicates to plot MTT assay.
Wound healing assay
A 70% confluent T25 flask of HCT-15 cells was taken and it was seeded in 6-well plates. For cell seeding, media were removed from culture flask and 1 ml PBS was added to wash the toxins and later it was discarded. After the wash, 1 ml trypsin was added in the culture flask and kept in 5% CO2 Incubator at 37°C for 5 minutes for trypsination of cells which detaches them from the flask. To this 1ml of Complete RPMI 1640 was added to it and the flask was washed thoroughly to remove any left adherent cells. This cell suspension was centrifuged at 1500 rpm for 5 min. The supernatant was discarded and pellet was resuspended in complete media. 5x103 cells of HCT15 were seeded in 6 wells plate which was incubated for 24hrs in 5% CO2 Incubator at 37°C so that cells reaches their confluency level. After the cells were confluent, two parallel scratch wounds were made with the help of sterile 200 μl and the growth medium was removed and PBS was added to prevent killing of the cells. Then, quercetin was added to the wells (IC30). The wound was observed at 0 h, 24 h, and 30 h, and images were taken and the gap distances of the wounds were measured with the help of Magnus Analytical MagVision, Version: x64.
Matrix metalloproteinase-9 crystal structure and quercetin derivatives: Retrieval and their preparation
X-ray crystallographic structures of MMP-9 were retrieved from PDB (PDB ID: 4XCT) with 1.3 Š resolution. Crystal structure was then prepared by adding hydrogens, minimized pH 7.4 at which created disulfide bonds by using Protein Preparation wizard.
Quercetin derivatives were retrieved from the PubChem database by applying Lipinski's rule of five with a 95% similarity. All retrieved quercetin derivatives along with quercetin were subjected to molecular energy minimization using the OPLS-2005 force field to obtain energetically stable conformations.
The binding site of MMP-9 (PDB ID: 4XCT) was then evaluated by PDBsum, and Leu187, Leu188, Ala189, Leu222, Val223, His226, Glu227, His230, His236, Leu243, Tyr245, Pro246, Met247, and 248 were considered as binding site residues [Figure 1].
|Figure 1: Amino acid residues involved in binding pocket of matrix metalloproteinase-9 receptor|
Click here to view
In order to reveal the binding mechanism of quercetin and its derivatives with MMP-9 (PDB ID: 4XCT), molecular docking calculations were performed with FlexX software. The crystal structure of MMP-9 receptor was taken from the PDB database and was treated as rigid and quercetin, and its derivatives were treated as flexible to generate their possible conformation during the docking calculations. The binding site of the MMP-9 receptor was defined using selected residues [Figure 1] in receptor preparation wizard of FlexX software.
All energy-minimized molecules were then docked into MMP-9-binding cavity to predict possible conformations of the compounds. The docking parameter in FlexX was kept at its default value. A number of conformations were generated for each compound, and the best conformations with the highest docking score were selected for further analysis.
All data reported are the arithmetic mean from three independent experiments performed in triplicate ± standard deviation unless stated otherwise
| Results and Discussion|| |
Effect of quercetin on HCT-15 cell line
Cell proliferation assay was carried out on HCT-15 cell line with different concentrations of quercetin (10−4, 10−5, 10−6, and 10−7 M) at 24, 48, and 72 h. MTT reduction to insoluble formazan by mitochondrial dehydrogenase was used as a validity parameter. The results [Figure 2] showed that quercetin shows antiproliferative activity on HCT-15 cells in a time- and dose-dependent manner.
|Figure 2: Quercetin shows antiproliferative activity on HCT-15 cell line in a dose- and time-dependent manner|
Click here to view
Effect of quercetin on migration of HCT-15 cells (wound healing assay)
A number of evidence have shown that MMP-9 is expressed in numerous types of cancers. The inhibition of MMP-9 can reduce cancer cell migration. With this hypothesis, we treated colon cancer cells (HCT-15) with quercetin (IC30) for different time points to understand whether quercetin inhibits HCT-15 cell migration.
[Figure 3] shows that with quercetin treatment, cell migration was reduced. At 0 h, just after the wound formation, the width of the scratch is 850 μm, which subsequently reduced to 800 μm and 740 μm with quercetin treatment for 24 h and 30 h. There is a 1.14-fold inhibition as compared to initial wound, proving quercetin as a good candidate for colon cancer cell migration inhibition. However, less bioavailability of quercetin and search for more efficient small molecule analog of quercetin that can bind specifically to MMP-9, target, and disrupt the function of protein leaves a scope to explore more efficient inhibitors through in silico approach.
|Figure 3: Quercetin treatment prevents wound healing in HCT-15 cells. (a-c) Quercetin treatment. (d-f) Untreated|
Click here to view
Molecular interaction analysis of quercetin and its derivatives with matrix metalloproteinase-9
To analyze the binding interactions of the quercetin and its derivatives with MMP-9 receptor, we used the molecular docking approach using FlexX software. After docking calculations, all conformers of quercetin and its derivatives were ranked according to their docking score. Top five best docked conformers of quercetin derivatives were selected having a better affinity toward MMP-9 receptor than quercetin and subjected to analysis of the binding mode and molecular interaction in the MMP-9-binding cavity [Table 1].
|Table 1: Detailed molecular docking analysis of quercetin and its derivatives with matrix metalloproteinase-9|
Click here to view
Molecular interaction and docking calculations reveal that lead compound (PubChem CID: 25164913) gives binding energy −33.1489 kcal/mol and was found to be more effective against MMP-9 as MMP-9 inhibitor. In case of quercetin, the binding energy was −25.7250 kcal/mol, which is higher than lead compound (PubChem CID: 25164913) suggest, quercetin having less affinity toward MMP-9.
The molecular interaction study of quercetin and lead compound (PubChem CID: 25164913) gives insights to understand their binding mode with MMP-9 receptor and critical amino acids of MMP-9 which determine the potency of docked compounds. In case of lead compound (PubChem CID: 25164913), five hydrogen bonds formed with the Ala189, Gly186, Gly227, Tyr245, and Arg249 having bond distances of 1.97 Š, 2.11 Š, 1.61 Š, 2.07 Š, and 2.11 Š, respectively. Another two residues of MMP-9 receptor were involved in Pi-PI interactions with lead compound including His226 and tyr248 at 4.22 Š and 5.21 Š bond distance, respectively, which increases the binding stability of lead compound (PubChem CID: 25164913) with MMP-9 receptor [Figure 4].
|Figure 4: Binding pose of lead compound (PubChem CID: 25164913) with matrix metalloproteinase-9 receptor. Matrix metalloproteinase-9 receptor presents in ribbon, lead compounds shown in ball and stick model, and amino acid residues shown in tube model. Black-dashed lines indicate hydrogen bonds, purple-dashed lines indicate aromatic hydrogen bond, and cyan-dashed lines indicate pi-pi stacking|
Click here to view
[Figure 5] shows quercetin forms, four hydrogen bonds, two Pi-Pi stacking, and one aromatic hydrogen bond with MMP-9 receptor. The amino acid residues of MMP-9, Ala189, Gly186, Gly227, and Leu243 forming hydrogen bond with quercetin at 1.92 Š, 2.29 Š, 1.91 Š, and 1.80 Š bond distances, respectively. Other residues His226 and Arg249 contribute to PI-Pi stacking with quercetin at a bond distance of 4.28 Š and 5.26 Š, respectively. Aromatic hydrogen forms between Tyr245 and quercetin at bond distance of 2.79 Š. Furthermore, the superimposition of quercetin and lead compound (CID: 25164913) shows minimal conformational changes [Figure 6], suggesting that docking protocol is validated and the lead compound occupies the similar space with space occupied by the quercetin in the MMP-9-binding cavity.
|Figure 5: Binding pose of quercetin (PubChem CID: 25164913) with matrix metalloproteinase-9 receptor. Matrix metalloproteinase-9 receptor presents in ribbon, quercetin shown in ball and stick model, and amino acid residues shown in tube model. Black-dashed lines indicate hydrogen bonds, purple-dashed lines indicate aromatic hydrogen bond, and cyan-dashed lines indicate pi-pi stacking|
Click here to view
|Figure 6: Superimposition of quercetin and lead compound (CID: 25164913). Quercetin shown in red color with ball and stick model and lead compound shown in green color with ball and stick model|
Click here to view
| Conclusion|| |
Quercetin treatment on HCT-15 cells shows an antiproliferative and antimigratory activity in a time-dependent manner. Molecular docking analysis concludes that the PubChem compound (CID: 25164913) shows a better binding affinity toward MMP-9 receptor as compared to standard compound quercetin. The docking pose superimposition of quercetin and lead compound (CID: 25164913) validated docking protocol with minimal atomic displacements of both the compounds within binding cavity of MMP-9 receptor. The lead compound (CID: 25164913) from in silico study could be useful for designing and development of novel MMP-9 inhibitors.
The authors are thankful to Dr. D. Y. Patil Biotechnology and Bioinformatics Institute, Dr. D. Y. Patil Vidyapeeth for physical infrastructure. The in vitro experimentations of this study are supported by the DST-Early Career Research Award (ECR/2016/000943), Department of Science and Technology, Government of India, and Dr. D. Y. Patil Vidyapeeth seed grant (DPU/14/2016, dated 06/01/2016). The authors also acknowledge the Department of Science and Technology-Science and Engendering Research Broad (DST-SERB), Government of India (File No. YSS/2015/002035), for computational support to perform molecular docking calculation.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Ferlay J, Shin HR, Bray F, Forman D, Mathers C, Parkin DM. Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008. Int J Cancer 2010;127:2893-917.
Mohandas KM. Colorectal cancer in India: Controversies, enigmas and primary prevention. Indian J Gastroenterol 2011;30:3-6.
Miller KD, Siegel RL, Lin CC, Mariotto AB, Kramer JL, Rowland JH, et al
. Cancer treatment and survivorship statistics, 2016. CA Cancer J Clin 2016;66:271-89.
Mohandas KM, Desai DC. Epidemiology of digestive tract cancers in India. V. Large and small bowel. Indian J Gastroenterol 1999;18:118-21.
Sung JJ, Lau JY, Goh KL, Leung WK; Asia Pacific Working Group on Colorectal Cancer. Increasing incidence of colorectal cancer in Asia: Implications for screening. Lancet Oncol 2005;6:871-6.
Stetler-Stevenson WG, Yu AE. Proteases in invasion: Matrix metalloproteinases. Semin Cancer Biol 2001;11:143-52.
Cathcart J, Pulkoski-Gross A, Cao J. Targeting Matrix Metalloproteinases in Cancer: Bringing New Life to Old Ideas. Genes Dis 2015;2:26-34.
Yang B, Tang F, Zhang B, Zhao Y, Feng J, Rao Z. Matrix metalloproteinase-9 overexpression is closely related to poor prognosis in patients with colon cancer. World J Surg Oncol 2014;12:24.
Juric V, O'Sullivan C, Stefanutti E, Kovalenko M, Greenstein A, Barry-Hamilton V, et al
. MMP-9 inhibition promotes anti-tumor immunity through disruption of biochemical and physical barriers to T-cell trafficking to tumors. PLoS One 2018;13:e0207255.
Coussens LM, Fingleton B, Matrisian LM. Matrix metalloproteinase inhibitors and cancer: Trials and tribulations. Science 2002;295:2387-92.
Levin VA, Phuphanich S, Yung WK, Forsyth PA, Maestro RD, Perry JR, et al
. Randomized, double-blind, placebo-controlled trial of marimastat in glioblastoma multiforme patients following surgery and irradiation. J Neurooncol 2006;78:295-302.
Levin M, Udi Y, Solomonov I, Sagi I. Next generation matrix metalloproteinase inhibitors – Novel strategies bring new prospects. Biochim Biophys Acta Mol Cell Res 2017;1864:1927-39.
Magne Nde CB, Zingue S, Winter E, Creczynski-Pasa TB, Michel T, Fernandez X, et al
. Flavonoids, breast cancer chemopreventive and/or chemotherapeutic agents. Curr Med Chem 2015;22:3434-46.
Du G, Lin H, Yang Y, Zhang S, Wu X, Wang M, et al
. Dietary quercetin combining intratumoral doxorubicin injection synergistically induces rejection of established breast cancer in mice. Int Immunopharmacol 2010;10:819-26.
Banerjee K, Basu S, Das S, Sinha A, Biswas MK, Choudhuri SK. Induction of intrinsic and extrinsic apoptosis through oxidative stress in drug-resistant cancer by a newly synthesized Schiff base copper chelate. Free Radic Res 2016;50:426-46.
Schrödinger Release 2018-4: Schrödinger Release 2018-4 Protein Preparation Wizard. New York: Schrödinger, LLC; 2018.
Laskowski RA, Jabłońska J, Pravda L, Vařeková RS, Thornton JM. PDBsum: Structural summaries of PDB entries. Protein Sci 2018;27:129-34.
Kramer B, Rarey M, Lengauer T. Evaluation of the FLEXX incremental construction algorithm for protein-ligand docking. Proteins 1999;37:228-41.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]